1,2-dihydropyrido[3,4-b]pyrazines

ABSTRACT

1,2-Dihydropyrido[3,4-b]pyrazines are provided which possess anticancer activity. The compounds have the structure: ##STR1## wherein Y is CH 2  or N(CH 3 ); R 1  is a lower alkyl group; e.g., an alkyl group containing up to six carbon atoms such as methyl, ethyl, propyl, butyl, etc.; R 2  is a member selected from the group consisting of hydrogen, CH 3  O or Cl; and R 3  and R 4  are either both hydrogen or one is hydrogen and the other is a lower alkyl group.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part of application Ser. No.362,480, filed Mar. 26, 1982 now abandoned.

BACKGROUND OF THE INVENTION

This invention relates to novel 1,2-dihydropyrido[3,4-b]pyrazines, alsoknown as 1-deaza-7,8-dihydropteridines. This invention also relates to aprocess for making such compounds and to novel intermediates obtained insaid process.

The antimitotic chemical agents commonly known as spindle poisons areplant products of which the best known are colchicine, podophyllotoxin,and the vinca alkaloids. [L. Wilson, J. R. Bamburg, S. B. Mizel, L. M.Grisham and K. M. Creswell, Federation Proceedings, 33, 158 (1974)]. Twomembers of the latter, vincristine and vinblastine, are currently usedclinically in the treatment of neoplasms. Although these agents producea number of biochemical actions such as the inhibition of macromolecularsynthesis, their primary effect is to prevent mitosis by interferingwith the function of microtubules, which results in the accumulation ofcells in metaphase. In addition, several benzimidazol-2-yl carbamateshave been introduced as fungicides, anthelmintics and antitumoralagents. [L. C. Davidse and W. Flach, J. Cell Biol., 72, 174 (1977)].These compounds also prevent mitosis and their biological activity canprobably be attributed to interference with the formation or functioningof microtubules.

The development of procedures for the preparation of 1-deazapteridinesis reported by J. A. Montgomery and N. F. Wood, J. Org. Chem., 29, 734(1964); R. D. Elliott, C. Temple, Jr. and J. A. Montgomery, J. Org.Chem., 33, 533 (1968); R. D. Elliott, C. Temple, Jr., J. L. Frye and J.A. Montgomery, J. Org. Chem., 36, 2818 (1971); and R. D. Elliott, C.Temple, Jr. and J. A. Montgomery, J. Med. Chem., 17, 553 (1974). Thesereferences disclose the preparation and use of various1,2-dihydro[3,4-b]pyrazine derivatives. Thus, the 1964 J. Org. Chem.reference discloses the compounds: ##STR2## The 1968 J. Org. Chem.reference discloses the compound: ##STR3## The 1971 J. Org. Chem.reference discloses the compounds: ##STR4## The J. Med. Chem. referencediscloses that a dihydro-1-deazapteridine precursor of1-deazamethotrexate showed activity against leukemia L1210 in mice. Anabstract presented at the 28th Southeast Regional Meeting of theAmerican Chemical Society in Gatlinburg, Tenn., Oct. 27-29, 1976discloses that the compound ##STR5## showed cytotoxicity in the KB cellculture screen and activity against leukemia L1210 in mice.

An abstract of a paper by B. J. Bowdon, G. P. Wheeler, C. G. Temple andJ. A. Montgomery in AACR Abstracts, Vol. 22, March 1981 (page 25)discloses that a compound designated as "NSC-181928" was active againstseveral neoplasms. NSC-181928 is the designation for the compound##STR6##

SUMMARY OF THE INVENTION

It has now been found that certain 1,2-dihydropyrido[3,4-b]pyrazinespossess anticancer activity. The compounds of this invention have thestructure: ##STR7## wherein Y is CH₂ or N(CH₃); R₁ is a lower alkylgroup, e.g., an alkyl group containing up to six carbon atoms such asmethyl, ethyl, propyl, butyl, etc.; R₂ is a member selected from thegroup consisting of hydrogen, CH₃ O or Cl; and R₃ and R₄ are either bothhydrogen or one is hydrogen and the other is a lower alkyl group, e.g.,one containing 1 to 3 carbon atoms.

Compounds of Formula I wherein R₃ is hydrogen may be prepared byaminating a lower alkyl ester of6-amino-4-chloro-5-nitropyridin-2-ylcarbamate having the structure:##STR8## with the oxime of an alpha-amino ketone having the structure:##STR9## to give a compound having the structure: ##STR10## wherein R₁,R₂, R₄ and Y are the same as previously defined, further provided thatR₂ may be a nitro group. The compound of Formula IV is hydrolyzed, e.g.,by acid hydrolysis to give the corresponding ketone having the formula:##STR11## wherein R₁, R₂, R₄ and Y are the same as previously defined,further provided that R₂ may be a nitro group. The compound of Formula Vis converted to the compound of Formula I by catalytic hydrogenation. Anintermediate product formed during hydrogenation has the formula:##STR12## wherein R₁, R₂, R₄ and Y are the same as previously defined.

Compounds of Formula I wherein R₄ is hydrogen may be prepared byaminating the compound of Formula II with an alphaamino alcohol havingthe structure: ##STR13## to give a compound having the structure:##STR14## which is oxidized to give a ketone having the structure:##STR15## wherein R₁, R₂, R₃ and Y are the same as previously defined,further provided that R₂ may be a nitro group. The compound of FormulaVA is converted to a compound of Formula I by catalytic hydrogenation.

DETAILED DESCRIPTION OF THE INVENTION

The compounds of the invention form pharmaceutically acceptable saltswith both organic and inorganic acids. Examples of suitable acids forsalt formation are hydrochloric, sulfuric, phosphoric, acetic, citric,oxalic, malonic, salicyclic, malic, fumaric, succinic, ascorbic, maleic,methanesulfonic, and the like. The salts are prepared by contacting thefree base form with an equivalent amount of the desired acid in theconventional manner. The free base forms may be regenerated by treatingthe salt form with a base. For example, dilute aqueous base solutionsmay be utilized. Dilute aqueous sodium hydroxide, potassium carbonate,ammonia, and sodium bicarbonate solutions are suitable for this purpose.The free base forms differ from their respective salt forms somewhat incertain physical properties such as solubility in polar solvents, butthe salts are otherwise equivalent to their respective free base formsfor purposes of the invention.

Also embraced within the purview of the present invention aretherapeutic compositions of matter useful for ameliorating cancerdiseases in mammals and containing the 1-deaza-7,8-dihydropteridines ofthis invention or pharmaceutically acceptable salts thereof.

The active ingredients of the therapeutic compositions and the novelcompounds of the present invention inhibit transplanted mouse tumorgrowth when administered in amounts ranging from about 5 mg to about 200mg per kilogram of body weight per day. A preferred dosage regimen foroptimum results would be from about 5 mg to about 50 mg per kilogram ofbody weight per day, and such dosage units are employed that a total offrom about 350 mg to about 3.5 grams of the active compound for asubject of about 70 kg of body weight are administered in a 24-hourperiod. This dosage regimen may be adjusted to provide the optimumtherapeutic response. For example, several divided doses may beadministered daily or the dose may be proportionally reduced asindicated by the exigencies of the therapeutic situation. A decidedpractical advantage is that the active compound may be administered inany convenient manner such as by the oral, intravenous, intramuscular orsubcutaneous routes.

The active compounds may be orally administered, for example, with aninert diluent or with an assimilable edible carrier, or they may beenclosed in hard or soft shell gelatin capsules, or they may becompressed into tablets, or they may be incorporated directly with thefood of the diet. For oral therapeutic administration, the activecompounds may be incorporated with excipients and used in the form ofingestible tablets, buccal tablets, troches, capsules, elixirs,suspensions, syrups, wafers and the like. Such compositions andpreparations should contain at least 0.1% of active compound. Thepercentage of the compositions and preparations may, of course, bevaried and may conveniently be between about 2 and about 60% of theweight of the unit. The amount of active compound in suchtherapeutically useful compositions is such that a suitable dosage willbe obtained. Preferred compositions or preparations according to thepresent invention are prepared so that an oral dosage unit form containsbetween about 5 and about 200 milligrams of active compound.

The tablets, troches, pills, capsules and the like may also contain thefollowing: a binder such as gum tragacanth, acacia, corn starch orgelatin; excipients such as dicalcium phosphate; a disintegrating agentsuch as corn starch, potato starch, alginic acid and the like; alubricant such as magnesium stearate; and a sweetening agent such assucrose, lactose or saccharin may be added or a flavoring agent such aspeppermint, oil of wintergreen or cherry flavoring. When the dosage unitform is a capsule, it may contain, in addition to materials of the abovetype, a liquid carrier. Various other materials may be present ascoatings or to otherwise modify the physical form of the dosage unit.For instance, tablets, pills or capsules may be coated with shellac,sugar or both. A syrup or elixir may contain the active compound,sucrose as a sweetening agent, methyl and propylparabens aspreservatives, a dye and flavoring such as cherry or orange flavor. Ofcourse, any material used in preparing any dosage unit form should bepharmaceutically pure and substantially non-toxic in the amountsemployed. In addition, the active compounds may be incorporated intosustained-release preparations and formulations.

The active compounds may also be administered parenterally orintraperitoneally. Solutions of the active compound as a free base orpharmaceutically acceptable salt can be prepared in water suitably mixedwith a surfactant such as hydroxypropylcellulose. Dispersions can alsobe prepared in glycerol, liquid polyethylene glycols, and mixturesthereof and in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), suitable mixtures thereof andvegetable oils. The proper fluidity can be maintained, for example, bythe use of a coating such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompound in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredient into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and the freeze-dryingtechnique which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

As used herein, "pharmaceutically acceptable carrier" includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents and the like. The use ofsuch media and agents for pharmaceutically active substances is wellknown in the art. Except insofar as any conventional media or agent isincompatible with the active ingredient, its use in the therapeuticcompositions is contemplated. Supplementary active ingredients can alsobe incorporated into the compositions.

It is especially advantageous to formulate parenteral compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuitable as unitary dosages for the mammalian subjects to be treated;each unit containing a predetermined quantity of active materialcalculated to produce the desired therapeutic effect in association withthe required pharmaceutical carrier. The specification for the noveldosage unit forms of the invention are dictated by and directlydependent on (a) the unique characteristics of the active material andthe particular therapeutic effect to be achieved, and (b) thelimitations inherent in the art of compounding such an active materialfor the treatment of disease in living subjects having a diseasedcondition in which bodily health is impaired as herein disclosed indetail.

The principal active ingredient is compounded for convenient andeffective administration in effective amounts with a suitablepharmaceutically-acceptable carrier in dosage unit form as hereinbeforedisclosed. A unit dosage form can, for example, contain the principalactive compound in amounts ranging from about 0.1 to about 400 mg, withfrom about one to about 30 mg being preferred. Expressed in proportions,the active compound is generally present in from about 0.1 to about 400mg/ml of carrier. In the case of compositions containing supplementaryactive ingredients, the dosages are determined by reference to the usualdose and manner of administration of the said ingredients.

A preferred lower alkyl ester of6-amino-4-chloro-5-nitropyridin-2-ylcarbamate is the ethyl ester, i.e.,ethyl 6-amino-4-chloro-5-nitropyridin-2-ylcarbamate. This compound isprepared according to the procedure described by R. D. Elliott, C.Temple, Jr. and J. A. Montgomery, J. Org. Chem., 31, 1890 (1966).

Oximes of alpha-amino ketones, i.e., compounds of Formula III, may beprepared by known prior art procedures. Thus, they can be prepared bycondensing the corresponding alpha-amino ketones with hydroxylaminehydrochloride in a refluxing mixture of pyridine and ethanol to give theoxime derivatives [R. D. Elliott, C. Temple, Jr. and J. A. Montgomery,J. Org. Chem., 35, 1676 (1970)].

The compounds of Formula III can also be prepared by alkylation ofphthalimide with the corresponding alpha-bromo-ketone, treatment of thealpha(phthalimido)-ketone product with hydroxylamine, and removal of thephthaloyl protecting group from the resulting oxime with hydrazine [R.D. Elliott, C. Temple, Jr. and J. A. Montgomery, J. Org. Chem., 35, 1676(1970)].

Examples of these two procedures for the preparation of compounds ofFormula III follow:

METHOD I 1-Amino-3-[(N-methyl-N-phenyl)amino]propanone Oxime

A mixture of 1-bromo-3-(phthalimido)propanone (145 g, 514 mmol),N-methylaniline (55.1 g, 514 mmol) and NaHCO₃ (43.2 g, 514 mmol) inN,N-dimethyl acetamide (1450 ml) was stirred at room temperature for 24hours followed by heating at 40° C. for 2 hours. The mixture was cooledin an ice bath and slowly diluted with cold water (440 ml). The yellowsolid that precipitated was collected by filtration, washed with a 1:3mixture of water-N,N-dimethyl acetamide (160 ml) and cold water, anddried in vacuo over P₂ O₅ : yield, 130 g. Dilution of the filtrate withadditional water (1200 ml) gave a second crop of product: yield, 20.1 g.The combined crops (150 g) were recrystallized from ethanol to give thediaminopropanone in 3 crops: yield, 145 g. A solution of this product(474 mmol), hydroxylamine hydrochloride (49.3 g, 709 mmol) and pyridine(482 ml) in ethanol (200 ml) was refluxed for 2.5 hours and evaporatedto dryness in vacuo. The residue was washed with cold water (2×500 ml),ethanol (250 ml) and recrystallized from ethanol to give the product in3 crops: yield, 143 g. A solution of the resultant oxime (442 mmol) inethanol (5300 ml) at 70° C. was treated dropwise during 20 minutes witha solution of 95% hydrazine (16.4 g) in ethanol (200 ml). The resultingsolution was heated at 40° C. for 22 hours, and the cooled reactionmixture was treated with 1N HCl (485 ml). After stirring in an ice bathfor 1 hour, the precipitated phthalhydrazide was removed by filtrationand washed with 1:1 ethanol-water (600 ml). The combined filtrate andwash was evaporated to dryness in vacuo (40° C.), the residue wasstirred with warm water (1500 ml), and after cooling, the insolubleyellow solid was removed by filtration and washed with water (200 ml).The clear yellow aqueous filtrate was treated with concentrated NH₄ OH(35 ml), and the oil that separated was extracted with CHCl₃ (3×300 ml).The combined extracts were dried over Na₂ SO₄ and evaporated to give agum, which solidified on drying in vacuo over P₂ O₅. The solid waspulverized, washed by vigorous stirring with cold water (700 ml), andredried in vacuo over P₂ O₅ to give the α-aminoketone oxime as a mixtureof syn and anti isomers: yield, 57.3 g.

METHOD II 1-Amino-4-phenyl-2-butanone Oxime

A solution of crude 1-amino-4-phenyl-2-butan-one hydrochloride (9.84 g,49.2 mmol) (Degraw, J., Isakotellis, P., Kisliuk, R., and Gaumont, Y.,J. Heterocyclic Chem., 1971, 8, 105), hydroxylamine hydrochloride (6.84g, 98.4 mmol) and sodium acetate. 3H₂ O (13.4 g, 98.4 mmol) in 50%ethanol (250 ml) was heated at 75°-80° C. for 30 minutes, filtered,treated with a hot solution of picric acid (11.7 g, 51.1 mmol), cooledto 25° C., filtered and allowed to stand for 2 days. The crystallinepicrate was collected, washed with 2:1 water-ethanol and dried in vacuo:yield 9.62 g, mp, 151° C. (Kofler Heizbank). The mother liquor wasevaporated to dryness in vacuo, and the residue was crystallized fromhot water (500 ml) to give an additional amount of the picrate: yield,3.62 g, mp 151° C. A solution of the picrate in 3:1 ethanol-water (400ml) was treated with washed BioRad AG1-X8 (Cl⁻) ion exchange resin (100g) and stirred for 18 hours. The solution was filtered and the resin waswashed with 3:1 ethanol-water. The filtrate and wash were treated withadditional resin (40 g), stirred for 2 hours and filtered. The almostcolorless solution was evaporated with ethanol (3×200 ml). The residuewas stirred with ethanol (100 ml), filtered and the precipitate wasrinsed with additional ethanol (40 ml). The filtrate and wash werediluted with diethyl ether (600 ml) to give a crystalline hydrochloridewhich was collected, washed with diethyl ether and dried in vacuo (P₂O₅): yield, 5.36 g.

Alpha-amino alcohols of Formula IIIA are prepared by the followingprocedure:

METHOD III 1-Amino-3-[[N-(4-chlorophenyl)-N-methyl]amino]2-propanol

A solution of epichlorohydrin (11 ml) and 4-chloro-N-methylaniline (11g, 78 mmol) in a mixture of ethanol (10 ml) and water (7 ml) wasrefluxed for 2 hours, diluted with water (20 ml), and extracted withdiethyl ether (3×50 ml). The combined extracts were evaporated todryness, the residue was treated with a solution of NaOH (5 g) in water(10 ml) for 1 hour, and the resulting mixture was extracted with diethylether (4×25 ml). The combined extracts were dried (MgSO₄) and evaporatedto dryness in vacuo to give1-[[N-(4-chlorophenyl)-N-methyl]amino]-2,3-epoxypropane: yield, 11 g(72%). A solution of this sample in a mixture of ethanol (50 ml) andliquid NH₃ (20 ml) was heated in a glass-lined stainless steel bomb at100° C. for 3 hours. The resulting reaction solution was evaporated todryness, and the dried residue was recrystallized from C₆ H₅ : yield,5.3 g (44%).

The oximes and alpha-amino alcohols set forth in Table I were preparedby Method I, Method II or Method III, as indicated in Table I. The firstcolumn of Table I sets forth the structure of the group ##STR16## inFormula III for the oximes prepared by Methods I and II and in FormulaIIIA for the alpha-amino alcohol prepared by Method III.

                                      TABLE I                                     __________________________________________________________________________    Alpha-Aminoketone Oximes (III) and Alpha-Aminoalcohols (IIIA)                                                            Analysis                                              Yield,                  Calcd, %  Found, %                 Compound.sup.a                                                                              Method                                                                             %.sup.b                                                                            M.p. °C.                                                                    Formula       C  H   N  C  H   N                 __________________________________________________________________________    C.sub.6 H.sub.5 N(CH.sub.3)CH.sub.2 (R.sub.4 = H)                                           I    56   c    C.sub.10 H.sub.15 N.sub.3 O.0.33H.sub.2                                                     60.30                                                                            7.93                                                                              21.09                                                                            60.43                                                                            7.61                                                                              21.01             4-CH.sub.3 OC.sub.6 H.sub.4 N(CH.sub.3)CH.sub.2                                             I    28   c    C.sub.11 H.sub.17 N.sub.3 O.0.37H.sub.2                                                     57.46                                                                            7.78                                                                              18.27                                                                            57.39                                                                            7.70                                                                              18.38             (R.sub.4 = H)                                                                 4-CH.sub.3 OC.sub.6 H.sub.4 N(CH.sub.3)CH.sub.2                                             I    27   141-8                                                                              C.sub.12 H.sub.19 N.sub.3 O.sub.2.2HCl.1.4H.s                                 ub.2 O.sup.d  42.96                                                                            7.17                                                                              12.53                                                                            43.39                                                                            7.19                                                                              12.49             (R.sub.4 = CH.sub.3)                                                          C.sub.6 H.sub.5 CH.sub.2 CH.sub.2 (R.sub.4 = H)                                             II   50   193  C.sub.10 H.sub.14 N.sub.2 O.HCl.0.3H.sub.2                                                  54.82                                                                            6.72                                                                              12.79                                                                            54.63                                                                            7.00                                                                              12.58             4-ClC.sub.6 H.sub.4 N(CH.sub.3)CH.sub.2                                                     III  32   .sup. 117-9.sup.e                                                                  C.sub.10 H.sub.15 ClN.sub.2 O                                                               55.95                                                                            7.04                                                                              13.05                                                                            56.29                                                                            7.18                                                                              13.17             (R.sub.4 = H)                                                                 __________________________________________________________________________     .sup.a R.sub.3 = H                                                            .sup.b Overall yield                                                          .sup.c Indefinite melting point                                               .sup.d Dihydrochloride salt was generated in ethanolic hydrogen chloride      and precipitated with ether                                                   .sup.e Resolidified and remelted at 123° C.                       

A compound of Formula II is aminated with a compound of Formula IIIunder nitrogen in refluxing ethanol containing triethylamine as an acidacceptor to give a compound of Formula IV. An example of this procedurefollows:

EXAMPLE 1 Ethyl6-Amino-4-[3-[(N-methyl-N-phenyl)amino]-2-oxopropylamino]-5-nitro-2-pyridinecarbamateOxime (IV: R₁ =C₂ H₅ ; R₂ =H; R₄ =H; Y=N(CH₃))

A solution of ethyl 6-amino-4-chloro-5-nitro-2-pyridinecarbamate (10.3g, 30.5 mmol), 1-amino-3-[(N-methyl-N-phenyl)propanone oxime (7.77 g,40.2 mmol) and triethylamine (4.27 g, 42.2 g, 42.2 mmol) in ethanol (200ml) was heated under N₂ at 75° C. for 24 hours. After cooling thereaction mixture the yellow solid was collected by filtration, washedwith cold ethanol, and dried in vacuo over P₂ O₅ at 65° C.: yield, 13.8g. The properties of the compound thus obtained are set forth in TableII.

Additional compounds were prepared similarly wherein the oxime ofExample 1 was replaced with other oximes. The properties of thesecompounds are set forth in Table II. The first column of Table II setsforth the structure of the group: ##STR17## in the startingalpha-aminoacetophenone oxime, see Formula III wherein R₁ is C₂ H₅ andR₄ is H or CH₃, and in the final product, see Formula IV, wherein R₁ isC₂ H₅ and R₄ is H or CH₃.

                                      TABLE II                                    __________________________________________________________________________    Ethyl 4-(Substituted)amino-6-amino-5-nitro-pyridine-2-carbamate Oximes                     Reaction                  Analyses                                            Time,                                                                              Yield,               Calcd, % Found, %                      Compound     Hours                                                                              %   M.p. °C.                                                                    Formula     C  H  N  C  H  N                       __________________________________________________________________________    C.sub.6 H.sub.5 N(CH.sub.3)CH.sub.2                                                        24   84  192-3 dec                                                                          C.sub.18 H.sub.23 N.sub.7 O.sub.5                                                         51.79                                                                            5.55                                                                             23.49                                                                            51.77                                                                            5.65                                                                             23.69                   (R.sub.4 = H)                                                                 4-CH.sub.3 OC.sub.6 H.sub.4 N(CH.sub.3)CH.sub.2                                            .sup. 15.sup.a                                                                     75  190  C.sub.19 H.sub.25 N.sub.7 O.sub.6                                                         51.00                                                                            5.63                                                                             21.91                                                                            50.97                                                                            5.89                                                                             21.87                   (R.sub.4 = H)                                                                 4-CH.sub.3 OC.sub.6 H.sub.4 N(CH.sub.3)CH.sub.2                                            16   77  --   C.sub.20 H.sub.27 N.sub.7 O.sub.6.2HCl.H.sub.2                                O.sup.b     43.48                                                                            5.66                                                                             17.75                                                                            43.53                                                                            5.30                                                                             17.65                   (R.sub.4 = CH.sub.3)                                                          C.sub.6 H.sub.5 CH.sub.2 CH.sub.2                                                          .sup. 24.sup.c                                                                     84  187  C.sub.18 H.sub.22 N.sub.6 O.sub.5                                                         53.72                                                                            5.51                                                                             20.89                                                                            53.61                                                                            5.56                                                                             20.77                   (R.sub.4 = H)                                                                 __________________________________________________________________________     .sup.a Solvent, methanol                                                      .sup.b Dihydrochloride salt prepared from a solution of the base in a         mixture of benzene and chloroform by the addition of ethanolic hydrogen       chloride                                                                      .sup.c Reaction temperature 54° C.                                

Treatment of a compound of Formula IV with a 1:1 mixture of 1Nhydrochloric acid and dioxane at 60° C. hydrolyzes the oxime function togive a compound of Formula V. An example of this procedure follows:

EXAMPLE 2 Ethyl6-Amino-4-[2-oxo-4-(phenylbutyl)amino]-5-nitro-2-pyridinecarbamate (V:R₁ =C₂ H₅ ; R₂ =H; R₄ =H; Y=CH₂)

A solution of the oxime of the title compound (6.17 g, 15.4 mmol) inwarm dioxane (60 ml) was treated with 1N HCl (120 ml), stirred at 55° C.for 1 hour and cooled in an ice bath. The precipitated hydrochloride wascollected, washed with cold water, then suspended in water (300 ml) andneutralized with 1N NaOH. The yellow product was collected, washed withwater and dried in vacuo (P₂ O₅): yield, 5.08 g (83%); m.p. 155° C.Anal. Calcd. for C₁₈ H₂₁ N₅ O₅ : C, 55.80; H, 5.46; N, 18.08. Found: C,55.42; H, 5.49; N, 18.12.

Amination of the compound of Formula II with the compound of FormulaIIIA in refluxing ethanol containing triethylamine as an acid acceptorgives a compound of Formula VII. Oxidation of a compound of Formula VIIgives a ketone of Formula VA. Two examples of this procedure follow:

EXAMPLE 3 A. Ethyl6-Amino-4-[3-[[N-(4-chlorophenyl)-N-methyl]amino]-2-hydroxypropylamino]-5-nitro-2-pyridinecarbamate(VII: R₁ =C₂ H₅ ; R₂ =4-Cl; R₃ =H; Y=N(CH₃))

A solution of ethyl 6-amino-4-chloro-5-nitro-2-pyridinecarbamate (10.0g), 1-amino-3-[[N-(4-chlorophenyl)-N-methyl]amino]-2-propanol (8.25 g),and triethylamine (10.7 ml) in methanol (120 ml) was heated at 60° C.for 18 hours and evaporated to dryness in vacuo. The dark residue waswashed with diethyl ether (1.5 l) to give a yellow solid. This solid waswashed with water (50 ml) and recrystallized twice from a mixture ofethanol and hexane: yield, 4.92 g. Similar treatment of the residueobtained from the ether wash from above gave a slightly impure sample ofproduct: yield, 5.89 g. Total yield, 10.81 g (64%); m.p., 181° C. Anal.Calcd. for C₁₈ H₂₃ ClN₆ O₅ : C, 49.26; H, 5.28; N, 19.15. Found: C,49.16; H, 5.46; N, 19.22.

B. Ethyl 6-Amino-4-[3-[[N-(4-chlorophenyl)-N-methyl]amino]-2-oxopropylamino]-5-nitro-2-pyridinecarbamate (VA: R₁ =C₂ H₅ ; R₂=4-Cl; R₃ =H; Y=N(CH₃))

A solution ofethyl-6-amino-4-[3-[[N-(4-chlorophenyl)-N-methyl]amino]-2-hydroxypropylamino]-5-nitro-6-pyridinecarbamate(1.76 g) and acetic anhydride (8 ml) in dimethyl sulfoxide (40 ml) wasstirred at room temperature for 20 hours, diluted with water (200 ml),and neutralized to pH 5.2 with 1N NaOH. The solid that deposited wascollected by filtration and dissolved in CHCl₃. Evaporation of thissolution to dryness and trituration of the resulting solid successivelywith water and ethanol gave the product: yield, 0.36 g (20%); m.p.123°-5° C. Anal. Calcd. for C₁₈ H₂₁ ClN₆ O₅.0.5H₂ O: C, 48.49; H, 4.97;N, 18.85. Found: C, 48.74; H, 4.65; N, 18.89.

EXAMPLE 4 A. Ethyl6-Amino-4-[3-[(N-methyl-N-phenyl)amino]-2-hydroxyoxopropylamino]-5-nitro-2-pyridinecarbamate(VII: R₁ =C₂ H₅ ; R₂ =H; R₃ =H; Y=N(CH₃))

This compound was prepared by the procedure described in Example 3A,substituting 1-amino-3-[(N-methyl-N-phenyl)amino]-2-propanol [O. Eisleb,German Pat. No. 473,219 (1926); Chem. Zentra., 100 (II), 350 1929)] for1-amino-3-[[N-(4-chlorophenyl)-N-methyl]-amino]-2 propanol: yield, 73%;m.p., 88°-90° C. Anal. Calcd. for C₁₈ H₂₄ N₆ O₅ : C, 53.46; H, 5.98; N,20.78. Found: C, 53.63; H, 5.93; N, 20.81.

B. Ethyl6-Amino-4-[3-[(N-methyl-N-phenyl)amino]-2-oxopropylamino]-5-nitro-2-pyridinecarbamate(VA: R₁ =C₂ H₅ ; R₂ =H; R₃ =H; Y=N(CH₃))

Crystalline o-phosphoric acid (3.08 g, 31.5 mmol) was added to a stirredsolution of ethyl6-amino-4-[3-[(N-methyl-N-phenyl)amino]-2-hydroxypropylamino]-5-nitro-2-pyridinecarbamate(3.18 g, 7.87 mmol) and N,N'-dicylohexylcarbodiimide (4.86 g, 23.6 mmol)in dry dimethyl sulfoxide (40 ml). The mildly exothermic reaction waskept below 25° C. by water bath cooling. After 2.5 hours, the deposit ofdicyclohexylurea was filtered off and washed with dimethyl sulfoxide (25ml). The filtrate was cooled in an ice bath and diluted slowly withwater (100 ml) to precipitate the product as a bright yellow solid thatwas washed thoroughly with water and dried in vacuo over P₂ O₅ : yield,2.89 g (80%); m.p., ˜80° C. with presoftening. Anal. Calcd. for C₁₈ H₂₂N₆ O₅.H₂ O.0.5(CH₃)₂ SO: C, 49.67; H, 5.92; N, 18.29. Found: C, 49.82;H, 5.69; H, 18.06.

The catalytic hydrogenation of a compound of Formula V or VA with athree-fold amount of Raney nickel in a large volume of ethanol (i.e.,more than one liter per gram) at atmospheric pressure at roomtemperature or with intermittent warming (e.g., to no higher than 60°C.) with a water bath gives the intermediate compound of Formula VIwhich is cyclized in situ with the elimination of water to give acompound of Formula I. Such reaction is shown in Example 5. Thecompounds of Formula I can also be prepared directly by hydrogenation ofa compound of Formula IV in the presence of Raney nickel as shown inExamples 6 and 7.

EXAMPLE 5 Ethyl5-Amino-1,2-dihydro-3-(2-phenylethyl)pyrido(3,4-b)pyrazine-7-carbamate(I: R₁ =C₂ H₅ ; R₂ =4-CH₃ O; R₃ =H; R₄ =H; Y=CH₂)

A solution of ethyl6-amino-4-amino-4-[2-oxo-4-(phenylbutyl)amino]-5-nitro-2-pyridinecarbamate(300 mg, 0.775 mmol) in N,N'-dimethyl acetamide (7 ml) was hydrogenatedin the presence of Raney nickel (890 mg, weighed wet, washed withethanol) for 20 hours to give an H₂ uptake of 58 ml (3.07 mmol). Thereaction mixture was filtered under N₂ and evaporated in vacuo at 25° C.The residual syrup was stirred with water (10 ml) to give a white powderwhich was collected, washed with water and dried in vacuo (P₂ O₅):yield, 230 mg. The properties are set forth in Table III.

EXAMPLE 6 Ethyl5-Amino-1,2-dihydro-3-[(N-methyl-N-pheny)aminomethyl]pyrido[3,4-b]pyrazine-7-carbamate(I: R₁ =C₂ H₅ ; R₂ =H; R₃ =H; R₄ =H; Y=N(CH₃))

A suspension of the oxime of ethyl6-amino-4-[3-[(N-methyl-N-phenyl)amino]-2-oxopropylaminol]-5-nitro-2-pyridinecarbamate(30.0 g, 71.9 mmol) and Raney nickel (60 g, weighed wet, washed withethanol) in ethanol (3000 ml) was hydrogenated at room temperature andatmospheric pressure with vigorous stirring. At the end of 12 hours, thehydrogen (7048 ml) absorbed corresponded to 134% of the theoretical for4 molar equivalents. The resulting mixture was heated nearly to boilingunder an atmosphere of N₂, and the catalyst was removed by filtrationand washed with boiling ethanol (5×200 ml). The combined filtrate andwash were concentrated to about 1000 ml in vacuo and cooled in an icebath to deposit the product as a pale yellow crystalline solid: yield,16.7 g. The properties are set forth in Table III.

EXAMPLE 7 Ethyl5-Amino-1,2-dihydro-3-[[N-(4-methoxyphenyl)-N-methylamino]methyl]-2-methylpyrido[3,4-b]pyrazine-7-carbamate[I: R.sub. 1 =C₂ H₅ ; R₂ =4-CH₃ O; R₃ =H; R₄ =CH₃ ; Y=N(CH₃)]

A suspension of the oxime of ethyl6-amino-4-[3-[[N-(4-methoxyphenyl)-N-methyl]amino]-1-methyl-2-oxopropylamino]-5-nitro-2-pyridinecarbamate(500 mg, 1.08 mmol) and Raney nickel (1.8 g, weighed wet, washed withethanol) in ethanol (500 ml) was hydrogenated at room temperature andatmospheric pressure. After 48 hours, thin layer chromatography showedthe absence of the oxime starting material. The catalyst was removed byfiltration, and the filtrate was concentrated in vacuo to 1/4 volume.The resulting solution was diluted with an equal volume of deoxygenatedwater to deposit the product: yield, 138 mg.

The compounds set forth in Table III were prepared by the procedure ofExample 5, Example 6 or Example 7, as indicated in Table III. Theproperties of these compounds are set forth in Table III. The firstcolumn of Table III sets forth the structure of the group: ##STR18## inthe starting material, see Formulas IV, V and VA wherein R₂ is C₂ H₅, R₃is hydrogen and R₄ is hydrogen or methyl, and in the final product, seeFormula I wherein R₁ is C₂ H₅, R₃ is hydrogen and R₄ is hydrogen ormethyl.

                                      TABLE III                                   __________________________________________________________________________    Ethyl 3-Substituted 5-Amino-1,2-dihydropyrido-[3,4-b]pyrazine-7-carbamates                 Procedure                                                                           Reaction                Analyses                                        of    Time,                                                                              Yield,             Calcd, %  Found, %                 Compound     Example                                                                             Hours                                                                              %   M.p. °C.                                                                    Formula   C  H   N  C  H   N                 __________________________________________________________________________    C.sub.6 H.sub.5 N(CH.sub.3)CH.sub.2                                                        6     12   63  .sup. 165.sup.a                                                                    C.sub.18 H.sub.22 N.sub.6 O.sub.2.                                                      59.94                                                                            6.46                                                                              22.92                                                                            59.98                                                                            7.05                                                                              22.92             (R.sub.4 = H)                    0.3H.sub.2 O.0.15C.sub.2 H.sub.6 O           C.sub.6 H.sub.5 N(CH.sub.3)CH.sub.2                                                        5     42   32  .sup.  80.sup.b                                                                    C.sub.18 H.sub.22 N.sub.6 O.sub.2.                                                      60.46                                                                            6.68                                                                              22.26                                                                            60.42                                                                            6.45                                                                              22.67             (R.sub.4 = H)                    0.5C.sub.2 H.sub.6 O                         4-ClC.sub.6 H.sub.4 N(CH.sub.3)CH.sub.2                                                    5     20   16  a    C.sub.18 H.sub.21 ClN.sub.6 O.sub.2.                                                    54.22                                                                            5.99                                                                              19.97                                                                            54.06                                                                            5.96                                                                              19.83             (R.sub.4 =  H)                   0.5H.sub.2 O.0.5C.sub.2 H.sub.6 O            4-CH.sub.3 OC.sub.6 H.sub.4 N(CH.sub.3)CH.sub.2                                            6      3   73  187  C.sub.19 H.sub.24 N.sub.6 O.sub.3                                                       59.36                                                                            6.29                                                                              21.86                                                                            59.16                                                                            6.29                                                                              21.88             (R.sub.4 = H)                                                                 4-CH.sub.3 OC.sub.6 H.sub.4 N(CH.sub.3)CH.sub.2                                            7     48   31  c    C.sub.20 H.sub.26 N.sub.6 O.sub.3.                                                      59.62                                                                            6.83                                                                              20.25                                                                            59.59                                                                            6.86                                                                              20.15             (R.sub.4 = CH.sub.3)             0.15H.sub.2 O.0.3C.sub.2 H.sub.6 O           C.sub.6 H.sub.5 CH.sub.2 CH.sub.2                                                          5     20   88  163  C.sub.18 H.sub.21 N.sub.5 O.sub.2                                                       63.70                                                                            6.24                                                                              20.64                                                                            63.35                                                                            6.25                                                                              20.47             (R.sub.4 = H)                                                                 __________________________________________________________________________     .sup.a prior sintering;                                                       .sup.b sintering;                                                             .sup.c indefinite                                                        

The 1,2-dihydropyrido[3,4-b]pyrazines of this invention are powerfulinhibitors of the proliferation of cultured lymphoid luekemia L1210cells as shown in Table IV. The concentration causing a 50% inhibitionof proliferation of the cells during 24 hours is similar to thatobserved for vincristine, vinblastine, and colchicine. Also, theaddition to the test medium of inosine, thymidine, glycine, citrovorumfactor, individually and in combinations, and elevated concentrations ofamino acids and vitamins did not overcome the inhibitions.

In addition to cell cytotoxicity, the 1,2-dihydropyrido[3,4-b]pyrazinesshowed activity against lymphocytic leukemia P388 cells (10⁶) implantedintraperitoneally in mice. Ethyl5-amino-1,2-dihydro-3-[(N-methyl-N-phenyl)aminomethyl]pyrido[3,4-b]pyrazine-7-carbamateand ethyl5-amino-1,2-dihydro-3-[[(N-(4-methoxyphenyl)-N-methyl]aminomethyl]pyrido[3,4-b]pyrazine-7-carbamateare also active in mice against P388 cells that were resistant tovincristine.

The 1,2-dihydropyrido[3,4-b]pyrazines of this invention atconcentrations that prevented any increase in the cell number during a24 hour period had little effect upon the synthesis of DNA, RNA, andprotein by cultured L1210 cells during exposure for four hours. Thisresult and those described above led to the determination of the effectof the 1,2-dihydropyrido[3,4-b]pyrazines upon cell division. Exposure ofcultured L1210 cells to the 1,2-dihydropyrido[3,4-b]pyrazines inhibitedcell division as measured by the mitotic index (MI) (Table IV), which isthe fraction of the cell population that is made up of metaphase cells.Subsequent experiments showed that these agents caused the accumulationin metaphase of human epidermoid carcinoma #2 cells, P388 cells, andP388 cells resistant to vincristine grown in suspension culture and ofcolon tumor #26 cells and colon tumor #38 cells grown on plasticsurfaces.

Table IV sets forth biological data for 1-deaza-7,8-dihydropteridines ofthis invention and for two prior art compounds. The first column ofTable IV sets forth the structure of the group R in the formula in theheading of the table for the 1-deaza-7,8-dihydropteridines tested andthe meaning of R.

The data in Table IV shows that the 1-deaza7,8-dihydropteridines of thisinvention are active against leukemia in laboratory animals.

                                      TABLE IV                                    __________________________________________________________________________    Biological Data - 1-Deaza-7,8-dihydropteridines                                ##STR19##                                                                                                         P388(c) 10.sup.6 Tumor                                                        cell implant, i.p.                                        L1210(a)                                                                              Mitotic Index(b)                                                                          Schedule,                                Compound         ID.sub.50 μM                                                                       12 h (μM)                                                                        24 h (μM)                                                                        Days % ILS (mg/kg)                       __________________________________________________________________________    Nocodazole       27 × 10.sup.-3                                                                        0.19(0.3)                                      Vincristine       1 × 10.sup.-3                                                                        0.62(0.3)                                                                           1    100(2.7)                            R = C.sub.6 H.sub.5 N(CH.sub.3)CH.sub.2                                                         8.4 × 10.sup.-3 (d)                                                             0.77(0.03)                                                                         0.80(0.3)                                                                           1    114(100)                            (R.sub.4 = H)                        1(e) 66(100)(f)                                                               1(g) 90(67)                              R = 4-CH.sub.3 OC.sub.6 H.sub.4 N(CH.sub.3)CH.sub.2                                             7.9 × 10.sup.-3 (h)                                                             0.64(0.03) 1    80(50)(i)                           (R.sub.4 = H)            0.49(0.3)   1    133(25)                                                                  1(g) 150(25)                             R = 4-CH.sub.3 OC.sub.6 H.sub.4 N(CH.sub.3)CH.sub.2                                            5.5 × 10.sup.-3                                                                 0.66(0.3)   1-5  53(48)                              (R.sub.4 = CH.sub.3)                                                          R = 4-ClC.sub.6 H.sub.4 N(CH.sub.3)CH.sub.2                                                    14.5 × 10.sup.-3                                                                 0.71(0.03) 1    65(100)                             (R.sub.4 = H)                                                                 R = C.sub.6 H.sub.5 CH.sub.2 CH.sub.2                                                          13 × 10.sup.-3                                                                        0.33(0.3)                                                                           1    29(100)                             (R.sub.4 = H)                                                                 R = 4-CH.sub.3 O.sub.2 CC.sub.6 H.sub.4 N(CH.sub.3)CH.sub.2 (j)                                58 × 10.sup.-3                                                                  0.61(0.3)   1-9  30(25)                              (R.sub.4 = H)            0.44(0.1)                                            __________________________________________________________________________     (a)Concentration of agent that inhibits proliferation of cultured lymphoi     leukemia L1210 cells to 50% control growth during 48 hours. G. P. Wheeler     B. J. Bowdon, J. A. Werline, D. J. Adamson and C. Temple, Jr., Cancer         Res., 42, 791 (1982).                                                         (b)Fraction of the cell population of cultured lymphoid leukemia L1210        cells in mitosis [ ref in a].                                                 (c)Lymphocytic leukemia P388. R. I. Geran, N. H. Greenbert, M. M.             MacDonald, A. M. Schumacker, and B. J. Abbott, Cancer Chemother. Rep., 3      (2) (1972).                                                                   (d)Average of 2determinations.                                                (e)Methotrexate-resistant line of P388 (designated tumor P7 by the Drug       Evaluation Branch, National Cancer Institute). In mice with 10.sup.6 cell     implant (IP), methotrexate at a dose of 2 mg/kg on the qd 1-9 schedule        gave a 2log cell kill against the sensitive line of P388 and a 2log           increase in cells against the methotrexateresistant line of P388.             (f)1-cure.                                                                    (g)Vincristine-resistant line of P388. L. J. Wilkoff and E. A. Dulmadge,      J. Natl. Cancer Inst., 61, 1521 (1978).                                       (h)Average of 2determinations.                                                (i)2-cures.                                                                   (j)R. D. Elliott, C. Temple, Jr., J. L. Frye, and J. A. Montgomery, J.        Org. Chem., 36, 2818 (1971).                                             

We claim:
 1. A 1,2-dihydropyrido[3,4-b]pyrazine having the formula:##STR20## wherein Y is CH₂ or N(CH₃); R₁ is a lower alkyl group; R₂ is amember selected from the group consisting of hydrogen, CH₃ O or Cl; R₃and R₄ are either both hydrogen or one is hydrogen and the other is alower alkyl group; and pharmaceutically acceptable salts thereof.
 2. Acompound as defined in claim 1 wherein R₁ is ethyl.
 3. Ethyl5-amino-1,2-dihydro-3-[[N-(4-methoxyphenyl)-N-methylamino]methyl]-2-methylpyrido[3,4-b]pyrazine-7-carbamate.4. Ethyl5-amino-1,2-dihydro-3-[(N-methyl-N-phenyl)-aminomethyl]pyrido[3,4-b]pyrazine-7-carbamate.5. Ethyl5-amino-1,2-dihydro-3-[[N-(4-methoxyphenyl)-N-methyl]aminomethyl]pyrido[3,4-b]pyrazine-7-carbamate.6. Ethyl5-amino-1,2-dihydro-3-[[N-(4-chlorophenyl)-N-methyl[aminomethyl]pyrido[3,4-b]pyrazine-7-carbamate.7. Ethyl5-amino-1,2-dihydro-3-(2-phenylethyl)pyrido[3,4-b]pyrazine-7-carbamate.8. A pharmaceutical composition in dosage unit form comprising aneffective amount to ammeliorate cancer diseases in mammals of a compoundas defined by claim 1 in association with a pharmaceutical carrier.